Hydrolysate of pichia minuta and cosmetic use thereof for controlling hair loss and stimulating regrowth

ABSTRACT

A cosmetic active principle including a hydrolysate of Pichia minuta comprising at least peptides. Additionally, the use of the cosmetic active principle including a hydrolysate of Pichia minuta comprising at least peptides for controlling hair loss and stimulating regrowth. Also, compositions containing same and a cosmetic hair treatment method.

RELATED APPLICATIONS

This application is a national phase application of InternationalApplication No. PCT/EP2018/050462 filed on Jan. 9, 2018 which claimspriority to French Application No. 1,770,024 filed on Jan. 9, 2017, theentity of both of which are incorporated herein by reference.

BACKGROUND

This invention relates to a particular cosmetic active ingredientobtained by yeast hydrolysis and its use for fighting hair loss andpromoting regrowth.

In a society that places importance on appearance, hair is a symbol ofbeauty and strength whose condition has a significant impact on one'sself-esteem and image. The acceleration of hair loss, scientificallyknown as alopecia, is perceived as a sign of early aging and candramatically affect the quality of life of those affected.

This is why many cosmetic products aimed at slowing hair loss have beenproposed, with a significant increase over the last ten years.

Alopecia is a common disorder with its androgenetic form being the mostcommon. It affects up to 30% of men under 30 and more than 50% of menover age 50. This progressive disorder, which is not a disease (Prie B.et al., “Oxidative stress in androgenetic alopecia”, Journal of Medicineand Life Vol. 9, Issue 1, January-March 2016, pp. 79-83), ischaracterized by the appearance of finer, shorter and non-pigmented hairwhich turns to vellus hair. It is caused by two main factors, geneticand hormonal, which cause hypersensitivity to a hormone:dihydrotestosterone (DHT). The latter is responsible for all biologicalchanges associated with this disorder. In addition, certainenvironmental parameters may also promote the appearance of androgeneticalopecia (AGA). This is the case with repeated use of aggressive hairproducts (dyes and other chemical agents), smoking or UV exposure.

Retention of thick hair is the result of equilibrium between hair lossand regrowth. Losing hair is a natural phenomenon, with an individuallosing between 50 and 125 every day. Thanks to a process ofregeneration, a constant renewal thereof is ensured: the so-called lifecycle of the hair or hair growth cycle. It is divided into three phases:

-   -   anagen (growth);    -   catagen (degeneration);    -   telogen (latency).

The hair or hair follicle consists of two main elements. The dermalpapilla is the driving force of its growth and lies at the base of thefollicle. It is composed of fibroblasts able to emit growth signals.These are then captured by the cells of the hair matrix. This structurethat surrounds the papilla is home to keratinocytes. In response tothese signals, they will proliferate to trigger the formation of a newhair stalk.

Hair growth is an energy-consuming process that requires functionalmitochondria in the cells, the morphological state of these dynamicorganelles conditioning energy production.

Other regulatory factors play a major role in the regeneration of hairand exhibit a malfunction during androgenetic alopecia:

-   -   the “signal” molecules, which allow the cells to communicate        with each other; within hair follicles, they orchestrate the        action of the fibroblasts of the papilla and keratinocytes of        the matrix to ensure hair growth;    -   epigenetics.

Currently, two chemical molecules are used in the treatment ofandrogenetic alopecia: minoxidil and finasteride. However, they areeffective in less than 50% of cases and have significant side effects(oily hair, dandruff, skin irritation, libido problems). There istherefore a real need for a new approach to promote hair growth whileavoiding the aforementioned problems.

In response, the invention proposes a natural cosmetic activeingredient, derived from yeast, able to act on three mechanisms forregulation of the regrowth of hair.

Several hydrolysates of yeast have already been used in cosmetics, suchas hydrolysates of Saccharomyces cerevisiae, Pichia anomala orTorulaspora delbrueckii. However, the invention relates to a hydrolysateobtained from a different yeast, Pichia minuta, which is very differentfrom already known yeasts used in cosmetics and which exhibits its owncharacteristics.

BRIEF SUMMARY

In particular, the invention relates to a cosmetic active ingredientincluding a hydrolysate of Pichia minuta, in particular a hydrolysate ofPichia minuta at least comprising peptides.

Advantageously, such an active ingredient is able to both stimulatemitochondrial dynamics for hair growth and correct expression defectsaffecting the “signal” and epigenetic molecules associated withandrogenetic alopecia. The cosmetic active ingredient of the inventionthus makes it possible to reactivate the dermal papilla and stimulategrowth of hair follicles.

As a consequence, the invention also relates to the non-therapeuticcosmetic use of such an active ingredient when applied to hair and/orscalp, for fighting hair loss and enable regrowth.

The invention also relates to cosmetic compositions, containing at least0.05% by weight, of the active ingredient of the invention, as well as anon-therapeutic cosmetic method for fighting hair loss and activatingits regrowth by application of such compositions to the hair or scalp.

DETAILED DESCRIPTION OF THE DRAWINGS

Other features and advantages will become apparent from the detaileddescription of the invention which follows, in which:

FIG. 1 represents the chromatographic profile (HPLC/RI) of thehydrolysate peptides of example 1,

FIG. 2 represents the aminogram of the peptides of the active ingredientof the invention of example 1,

FIG. 3 represents the chromatogram of the proteins of the three productspresented in the Comparative Test 1, obtained using the same processstarting for 3 different yeasts: Pichia minuta, Pichia heedii andSaccharomyces cerevisiae,

FIG. 4A represents an agar culture medium on which a biomass of Pichiaminuta was inoculated and incubated for 48 h at 30° C.,

FIG. 4B represents an agar culture medium on which a hydrolysate ofPichia minuta was inoculated and incubated for 48 h at 30° C.,

FIG. 5A represents an enlarged photo of the scalp before application ofa composition of the invention,

FIG. 5B shows a zoomed in picture of a scalp after 8 months ofapplication of a composition of the invention (point II.1),

FIG. 6A represents a photo of a scalp before application of acomposition of the invention, and,

FIG. 6B shows a picture of a scalp after 8 months of application of acomposition of the invention (point II.1)

DEFINITIONS

For the purposes of the invention, the term “cosmetic active agent” or“cosmetic active ingredient” means at least one molecule, preferentiallya set of molecules having an effect on the cells of the hair and/or thescalp, in particular on the fibroblasts of the dermal papilla and thecells of the hair matrix.

For the purposes of the invention, the terms “protein fraction” or“peptide compounds” of the hydrolysate of Pichia minuta means allproteins and peptides present in the hydrolysate of Pichia minuta.

For the purposes of the invention, the term “hydrolysate of Pichiaminuta” means any active ingredient derived from Pichia minuta yeast,obtained by a process comprising at least one step of enzymatic orchemical hydrolysis of Pichia minuta. The term hydrolysate of Pichiaminuta excludes molecules produced solely by fermentation of Pichiaminuta.

“Pichia minuta”, within the meaning of the invention, means any yeast ofthe Saccharomycetaceae family, of the Ogataea genus and Pichia minutaspecies. The Pichia minuta yeast has been registered in several yeastcollections, under numbers ATCC 26176, CBS 6511, MUCL 27758, MUCL 29976,DSM-70275, NRRL Y-411, NRRL Y-7953, NRRL Y-10948, Y-172, Y-2081, Y-2516,NCYC-499, 3622T, 9442, 3615T, NBRC 1473, NBRC 0975T, NBRC 10402, NBRC10746. It is also known by other names: Candida methanolovescens,Ogataea minuta, Ogataea nonfermentans. Pichia minuta can be isolatedfrom the azalea flower, Rhododendron indicum.

For the purposes of the invention, the term “atomization carrier” isunderstood to mean a neutral adjuvant added to a solution in order toproduce a powder during spray drying.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a cosmetic active ingredientincluding a hydrolysate of Pichia minuta.

The hydrolysate of Pichia minuta at least contains peptides. Preferably,the hydrolysate of Pichia minuta contains peptides which have amolecular weight of less than 3500 Da, preferably between 243 and 3500Da. These peptides play an important role in the effectiveness of theactive ingredient of the invention.

Preferably, the peptides which have a molecular weight of less than 3500Da represent at least 50% by weight of the protein fraction of thehydrolysate, still more preferably at least 80%.

In the hydrolysate, the protein fraction represents at least 40% byweight of the hydrolysate dry matter, preferably between 40 and 90%,more preferably between 50 and 70%.

The peptide compound content is preferentially determined by the LOWRYmethod (Lowry et al., Protein measurement with the folin reagent, J.Biol. Chem., 193, 265, 1951).

The amino acids which make up more than 5%, constituting the peptides ofthe hydrolysate (percent relative to the whole of the amino acidspresent in number) are preferably: alanine, arginine, aspartic acid,glutamic acid, leucine, lysine, serine, threonine, valine.

The aminogram of the peptides which make up the hydrolysate of theinvention can be obtained by ion exchange chromatography after acidhydrolysis of the sample.

The hydrolysate also preferably comprises sugars and/or minerals.

Preferably, the sugars represent at least 5% by weight of hydrolysatedry matter, in particular between 5 and 25%, more preferably between 10and 20%.

The sugar content of the hydrolysate can be determined by the DUBOISmethod (Dubois M et al., Analytical Chemistry, 28, 3, 350-356, 1956).

The minerals present in the hydrolysate are preferably calcium,potassium, sodium, chlorine and phosphorus. The analysis of the mineralsconstituting the ashes of the hydrolysate was carried out by opticalemission spectrometry (ICP/OES) and the measurement of chloride ions bytitration with silver nitrate.

Preferably, the ash content is between 5 and 35% by weight ofhydrolysate dry matter, more preferably between 20 and 30%.

The crude ash content can be determined by weighing the residuesresulting from the incineration of the samples of the active ingredientof the invention at 550° C., in an electric muffle furnace.

The hydrolysate of the invention can be obtained by chemical (acid orbase) or enzymatic hydrolysis. Preferably, an enzymatic hydrolysate isused.

The active ingredient of the invention can be in solid or liquid form.

When it is in liquid form, the active ingredient of the invention ispreferably the hydrolysate as described above. It is in the form of aclear liquid, with a weak odor and a slightly orangish yellow color.

When it is in solid form, the active ingredient of the invention ispreferably made up of the hydrolysate of Pichia minuta as previouslydescribed and of a carrier chosen from maltodextrin, gum arabic, soylecithin or isomalt. According to one particularly suitable production,the hydrolysate represents between 25 and 75% of the weight of theactive ingredient, preferably between 40 and 60%.

In the case of a solid form in which the active ingredient is associatedwith a carrier, the protein, sugar and ash contents in the activeingredient are modified, the carrier generally comprising mainly ofsugars. In the case where the atomization carrier is maltodextrin, in anamount between 25 and 75%, preferably between 40 and 60%:

the peptides represent at least 12%, in particular between 12 and 53% byweight of dry matter of the active ingredient, preferably between 20 and42%,

the sugars represent between 32 and 80% by weight of dry matter of theactive ingredient, preferably between 46 and 68%,

the ashes represent between 5 and 23% by weight of dry matter of theactive ingredient, preferably between 8 and 18%.

The hydrolysate constituting the active ingredient of the invention canbe obtained by any process comprising at least one step of hydrolysis ofPichia minuta, in particular a step for hydrolysis of the proteins.Preferably, it is obtained by a process involving a step of enzymatichydrolysis, in particular of enzymatic hydrolysis of the proteins, thatis to say a hydrolysis carried out enzymatically by means of proteolyticenzymes. This may be, for example, proteases of plant origin or derivedfrom microorganisms.

Prior to the process for obtaining the hydrolysate as such, the biomassof Pichia minuta should be produced. This step is carried out accordingto the culture method of the yeasts in a medium appropriate for theirdevelopment, in a manner conventional to those skilled in the art.

Once the biomass has been obtained, hydrolysis is carried out in orderto obtain active molecules. According to a particularly suitableembodiment, the active ingredient is obtained by the implementation ofthe following steps:

-   -   solubilization of the biomass of Pichia minuta in water    -   hydrolysis of proteins: hydrolysis conditions are chosen to        hydrolyze both the walls of the yeasts and the intracellular        environment of the yeasts to promote bioactive peptide        enrichment; preferably the hydrolysis is carried out        enzymatically by means of proteolytic enzymes, preferably-of        plant origin or derived from microorganisms;    -   inactivation of the enzyme(s), preferentially by heat treatment:        this inactivation is carried out according to the technical        recommendation of the supplier(s) of the enzyme(s);    -   separation of the soluble and insoluble phases, preferably by        centrifugation, and recovery of the soluble phase containing,        among other things, peptides and soluble proteins,    -   filtration to remove still suspended particles,    -   Purification by filtration to remove high molecular weight        molecules (enzymes and polymers . . . ), preferably molecules of        molecular weight greater than 5000 Da,    -   obtaining a filtrate, the hydrolysate of Pichia minuta, which        constitutes a first form of the active ingredient of the        invention, which is in liquid form.

The hydrolysate obtained at this stage may be further concentratedand/or purified in order to select the fractions of low molecularweight, preferably less than 3500 kDa, or even possibly less than 2000kDa, by successive ultrafiltration steps through varying porosityfilters, retaining the filtrates at each stage and/or by means of achromatographic type method, for example to specifically enrich thehydrolysate in these molecules.

The hydrolysate can then be dried and combined with a carrier to be insolid form. This phase can be achieved by implementing the followingsteps:

-   -   an atomization carrier, preferably maltodextrin, is added to the        hydrolysate of Pichia minuta, between 25 and 75% (mass/volume);    -   this solution is then concentrated under vacuum;    -   removal of bacteria is carried out by heat treatment;    -   atomization makes it possible to obtain a powder.

This presentation of the peptide hydrolysate corresponds to a highlyconcentrated form of active molecules, in particular, peptides.

The steps of the methods described above, taken individually, are commonin the field of extractions of active ingredients from natural rawmaterials and a person skilled in the art is able to adjust the reactionparameters based on their general knowledge.

The active ingredient of the invention is particularly effective againsthair loss and to activate its regrowth. In particular, the hydrolysateof Pichia minuta of the invention, when applied to the hair and/orscalp:

-   -   promotes mitochondrial dynamics, a key element for hair growth,    -   corrects the expression modifications of the “signal” molecules        appearing during androgenetic alopecia,    -   controls epigenetic actors during androgenetic alopecia, and    -   maintains dermal papilla activation and hair follicle growth        during androgenetic alopecia.

The growth of hair follicles is a process requiring a high energy input.Within the cell, it is the mitochondria that ensure energy production.For this, these organelles are able to adapt their morphology and,ultimately, their activity: mitochondrial dynamics This is based on twophenomena: fission and fusion. Fission is defined by the separation ofmitochondria which are then smaller and produce less energy. Conversely,fusion corresponds to the grouping of mitochondria in the form of anetwork. This relies notably on the intervention of mitofusin 1 (MFN1),a membrane protein involved in the fusion of the outer mitochondrialmembranes. This process allows them to intensify their metabolism andproduce more energy in the form of ATP.

Mitochondrial dynamics is involved in the biology of hair follicles. Inparticular, it is known that the morphology of mitochondria evolvestowards a fused form in dermal papilla cells. This phenomenon isaccompanied by an increase in mitochondrial enzymatic activity and theproduction of ATP. These mitochondrial changes provide the energyrequired to pass or maintain the growth phase of the hair cycle (Mifudeet al. “PDGF-AA-induced filamentous mitochondria benefit dermal papillacells in cellular migration”. International Journal of Cosmetic Science,37, 266-271 (2015)).

Advantageously, the active ingredient of the invention is able tostimulate the synthesis of mitofusin 1 in the fibroblasts of the dermalpapilla and thus to promote the growth of hair follicles.

In addition, the “signal” molecules present in the microenvironment ofthe dermal papilla are essential for hair growth. Three of them arederegulated in androgenetic alopecia:

-   -   IL-6 (Interleukin 6), which blocks the proliferation of the        cells of the matrix and inhibits the growth of the hair shaft;    -   DKK1 (Dickkopf 1), an inhibitor of the Wnt/β-catenin pathway,        which causes the apoptosis of keratinocytes of the follicle and        thus the entry into the catagen phase (latency) of the hair        cycle;    -   P16, a cell cycle inhibitor, which is responsible for the        premature senescence of fibroblasts of the dermal papilla.

In androgenetic alopecia, these three “signal” molecules areoverexpressed. Indeed, dermal papilla fibroblasts from alopecic areasshow an increase in P16 expression (Bahta A W et al. “PrematureSenescence of Balding Dermal Papilla Cells In Vitro Is Associated withP16^(INK4a) Expression” Journal of Investigative Dermatology, 128,1088-1094 (2008)), of the secretion of IL-6 (Kwack M H & al.“Dihydrotestosterone-Inducible Dickkopf 1 from Balding's Dermal PapillaCells Causes Apoptosis in Follicular Keratinocytes” Journal ofInvestigative Dermatology, 128, 262-269 (2008)) and the expression ofDKK1 (Fawzi M M T & al. “Assessment of tissue levels of dickkopf-1 inandrogenetic alopecia and alopecia areata.” Journal of CosmeticDermatology, 15, 10-15 (2015)). These deregulations are also observedfollowing treatment with DHT and may even be accentuated following theactivation of the androgen signaling pathway (Yang et al. “AndrogenReceptor Accelerates Premature Senescence of Human Dermal Papilla Cellsin Association with DNA Damage”. Plos One, 8, 1-10 (2013)).

Advantageously, the active ingredient of the invention is able to reducethe secretion of IL-6 and the expression of DKK1 and P16 in thefibroblasts of the dermal papilla. It thus makes it possible to correctthe abnormal expression of the “signal” molecules involved inandrogenetic alopecia.

In another aspect, epigenetics also plays a major role in the biology ofhair. Epigenetics studies the mechanisms that can regulate theexpression of genes in response to certain environmental stresses,without modifying the DNA pattern. All actors involved are grouped underthe name of epigenome. Among them, miRNAs hold an important place. Thesesmall RNA molecules are able to inactivate genes on the basis of theenvironment by modifying the stability of their target messenger RNAs.It has recently been demonstrated that miRNAs regulate various processessuch as proliferation and differentiation of dermal papilla fibroblastsand keratinocytes of the hair matrix. In addition, they regulateapoptosis during the catagen phase (Andl et al. “MicroRNAs (miRNAs) inthe control of HF development and cycling: the next frontiers in hairresearch” Experimental dermatology, 24, 821-826 (2015)).

In an alopecic context, the fibroblasts of the dermal papilla have aderegulated miRNA expression profile. These modifications includeoverexpression of miR-3663-3p and of let-7a-3p (Lee et al. “Analysis ofthe microRNA expression of normal human dermal papilla cells treatedwith 5α-dihydrotestosterone” Molecular Medicine Reports, 12, 1205-1212(2015)). Among the target genes for these miRNAs, some are involved inthe Wnt/β-catenin signaling pathway, an essential pathway for hairgrowth. It is activated following the binding of a ligand of the Wntfamily to its specific receptor and causes the accumulation ofβ-catenin. Ultimately, this leads to proliferation of dermal papillafibroblasts and induction of the hair growth phase (Ouji et al.“Maintenance of Dermal Papilla Cells by In Vitro Wnt-10b” Methods inMolecular Biology, 1516, 269-277 (2016)).

Advantageously, the active ingredient of the invention is able to limitthe expression of miRNA 3663-3p and of let-7a-3p in fibroblasts of thedermal papilla. Thus, it attenuates epigenetic deregulations associatedwith androgenetic alopecia.

It is also known that the dermal papilla is the control center for hairgrowth. The fibroblasts of the papilla emit signals to the keratinocytesof the hair matrix in order to regulate the development of the hair. Ina normal context, it takes place in a cycle having three phases:

-   -   anagen: hair growth phase over 2 to 5 years;    -   catagen: phase of degeneration lasting from a few days to a few        weeks;    -   telogen: latency phase for about 3 months.

In androgenetic alopecia, the alteration of the dynamics of the haircycle is a major deregulation. It results in an increase in theproportion of hair in the telogen phase (latency), the duration of whichis maintained or even lengthened. This is accompanied by a progressiveshortening of the anagen phase (growth), marked by a decrease inexpression of versican, a protein that is specific to this step (Soma etal. “Hair cycle-specific expression of versican in human hair follicles”Journal of Dermatological Science, 39, 147-154 (2005)). Thesederegulations as a whole are at the origin of the shrinking of hairfollicles to such an extent that the hair no longer reaches the surfaceof the skin.

The active ingredient of the invention is able to stimulate thesynthesis of versican in fibroblasts of the dermal papilla and thuspreserve the activation of the dermal papilla. In addition, it improvesthe growth of hair follicles and helps to maintain a significantsynthesis of versican and Ki-67. Thus, the active ingredient of theinvention makes it possible to reduce the lack of growth associated withandrogenetic alopecia by keeping hair follicles in the growth phase.

The invention thus relates to the use of an active ingredient of theinvention by means of application to the hair and/or scalp, for fightinghair loss and activate its regrowth, especially in people withandrogenetic alopecia. It acts on molecular abnormalities associatedwith androgenetic alopecia by correcting deregulation affecting thesignal molecules and epigenetics and by stimulating mitochondrialdynamics It is thus effective in normalizing two essential parametersfor hair development, namely: activation of the dermal papilla andstimulation of the growth of hair follicles.

The active ingredient of the invention is preferably used incompositions, these compositions comprising a cosmetically acceptablemedium. These are compositions in different galenic forms, suitable forapplication on the scalp and hair.

These compositions can, in particular, be in the form of oil-in-wateremulsions, water-in-oil emulsions, multiple emulsions (Water/Oil/Wateror Oil/Water/Oil), which can possibly be micro emulsions ornanoemulsions, or in the form of solutions, suspensions,hydrodispersions, aqueous gels or powders. They can be more or lessfluid and have the appearance of lotion, shampoo, cream or mousse.

It may be compositions comprising at least 0.05% of a hydrolysate ofPichia minuta of the invention, preferably between 0.5 and 10%.

These compositions comprise, on top of the active ingredient, aphysiologically acceptable and preferably cosmetically acceptablemedium, i.e. which does not cause unacceptable discomfort for the user,such as redness, pulling or tingling.

The compositions of the invention may contain as adjuvant at least onecompound chosen from:

-   -   oils, which may be chosen in particular from silicone oils,        whether linear or cyclic, volatile or non-volatile;    -   waxes, such as ozokerite, polyethylene wax, beeswax or carnauba        wax,    -   silicone elastomers,    -   surfactants, preferably emulsifiers, whether they are nonionic,        anionic, cationic or amphoteric,    -   co-surfactants, such as linear fatty alcohols,    -   thickeners and/or gelling agents,    -   humectants, such as polyols like glycerin,    -   dyes, preservatives, fillers,    -   tensors,    -   sequestering agents,    -   perfumes,    -   and their mixtures, without this list being limitative.

Examples of such adjuvants are specifically cited in the CTFA Dictionary(International Cosmetic Ingredient Dictionary and Handbook published bythe Personal Care Product Council).

Of course, a person skilled in the art will carefully select anycomplementary compounds, whether active or non-active, and theirquantity, so that the advantageous properties of the mixture are not,now or at a later stage, impaired by the planned addition.

These compositions are especially intended to be used for anti-hair lossand hair regrowth processes.

The invention thus also relates to a non-therapeutic method, namely anon-therapeutic cosmetic method for fighting hair loss and activatingits regrowth, which includes applying a composition which includes anactive ingredient of the invention to the hair and/or scalp, preferablyat least once a day for at least one month. Preferably, the compositionis a composition of the invention.

In order to illustrate these cosmetic effects for controlling hair lossand activation of the regrowth of hair, the following examples withtheir test results are presented.

EXAMPLES Example 1 Active Ingredient of the Invention in Liquid Form

The active ingredient according to Example 1 is obtained by implementingthe following steps:

-   -   solubilization of the Pichia minuta biomass in water at 50 g/l,    -   enzymatic hydrolysis with a proteolytic enzyme of bacterial        origin,    -   thermal inactivation, at 80° C., of the enzymatic activity,    -   centrifugation decantation to recover the supernatant,    -   filtration, and collection of the filtrate,    -   sterilizing filtration on 0.22μ filter.

The hydrolysate obtained is in the form of a clear liquid, of a slightlyorangish yellow color, with a weak odor.

It includes:

-   -   59% peptides by weight of dry matter, (determined by the LOWRY        method),    -   15% sugars by weight of dry matter, (determined by the DUBOIS        method),    -   26% ash by weight of dry matter, (determined by the weighing of        the residues resulting from the incineration of the samples of        the hydrolysate at 550° C. in an electric muffle furnace).

In addition, the peptides were characterized in order to determine theirmolecular weights and to quantify the various protein fractions by sizeexclusion FPLC chromatography.

The hydrolysate is analyzed under the following conditions:

-   -   Pump: FPLC ÄKTA (Pharmacia)    -   Column : Superdex Peptides TRICORN 10/300 GL (Pharmacia)    -   Mobile phase: 20 mM potassium phosphate buffer, pH 7.2 with 0.25        M NaCl.    -   Detector: UV at 280 nm    -   Flow: 0.5 mL/min    -   Injection volume: 200 μL

A calibration curve of the retention time of the markers as a functionof their molar mass is constructed (logarithmic fit). Table 1 summarizesthe retention times obtained for the different markers.

TABLE 1 Retention time of the different markers. Molecules Molecularweight (Da) Retention time (min) Cytochrome C 12,500 20.0 Aprotinin6,512 23.5 Vitamin B12 1,355 33.4 Cytidine 243 41.4

Thanks to the calibration curve, the following retention times could beestimated:

-   -   protein of 10,000 Da: retention time=21.4 min    -   protein of 3500 Da: retention time=25.2 min    -   protein of 2000 Da: retention time=30.3 min

Then, a fractionation program is constructed, on the basis of theretention times, in order to collect the following fractions(MM=molecular weight):

-   -   fraction 1: 10,000 Da<MM    -   fraction 2: 3,500 Da<MM<10,000 Da    -   fraction 3: 2,000 Da<MM<3,500 Da    -   fraction 4: 243 Da<MM<2,000 Da    -   fraction 5: MM<243 Da

The different fractions collected from the hydrolysate are quantified byspectrophotometric assay according to the LOWRY method (Lowry et al.,Protein measurement with the folie reagent, J. Biol. Chem., 193, 265,1951).

The chromatographic profile and the fractionation of the peptides of thehydrolysate are shown in FIG. 1 and the distribution of each fractiondetermined by spectrophotometric method is presented in Table 2.

TABLE 2 Distribution and Quantification of the Proteins of the MainActive Ingredient of Example 1 Molecular weight (Da) Division (%)Fraction 1: 10,000 Da < MM 0.3 Fraction 2: 3,500 Da < MM < 10,000 Da 2.9Fraction 3: 2,000 Da < MM < 3,500 Da 10.5 Fraction 4: 243 Da < MM <2,000 Da 83.3 Fraction 5: MM < 243 Da 3.0

More than 93% of peptides in the main active ingredient of Example 1 ofthe invention have molar masses of between 243 and 3500 Da. The amountof protein greater than 3500 Da represents less than 5% of the totalprotein.

An aminogram was also performed by HPLC chromatography of the hydrolysisof the product (concentrated hydrochloric acid for 24 hours) withdetection with ninhydrin and the results are shown in Table 3 and inFIG. 2.

TABLE 3 Aminogram carried out on the hydrolysate of Example 1 g/100 gDivision Alanine 2.32 9.6% Arginine 1.46 6.1% Aspartic acid 2.39 9.9%Cystine 0.19 0.8% Glutamic acid 4.99 20.9% Glycine 1.01 4.2% Histidine0.40 1.7% Isoleucine 1.07 4.5% Leucine 1.99 8.3% Lysine 1.86 7.7%Methionine 0.29 1.2% Phenylalanine 0.66 2.7% Proline 0.75 3.1% Serine1.33 5.5% Threonine 1.30 5.4% Tyrosine 0.60 2.5% Valine 1.45 6.0% Total24.06 100.0%

In addition, the ash distribution was determined after incineration at550° C. The analysis of the minerals was carried out by optical emissionspectrometry (ICP/OES) and the determination of chloride ions bytitration with silver nitrate.

It is presented in Table 4 below:

TABLE 4 Ash distribution of the hydrolysate of Example 1 Ashdistribution (%) Calcium 4 Potassium 18 Magnesium 1 Sodium 14 Chlorine43 Phosphorus 12 Sulfur 7 Not identified 1

Example 2 Active Ingredient of the Invention in Solid Form (Hydrolysateof Example 1+Maltodextrin)

The active ingredient of Example 1 is combined with maltodextrin toconstitute the active principle of Example 2.

It is obtained by implementing the following steps:

-   -   introduction of 50% of maltodextrin (mass) into the hydrolysate        of Example 1,    -   concentration until reaching a concentration factor of 5,    -   debacterialization 5 min at 95° C.,    -   atomization.

The active ingredient is in the form of a powder having a particle sizeof less than 500 μm, a weak odor, a slightly orangish yellow color.

It includes (determination according to the same methods as for thehydrolysate in Example 1):

-   -   28% peptides by weight of dry matter,    -   58% sugars by weight of dry matter,    -   14% ash by weight of dry matter.

Since maltodextrin includes only non-active sugars, the peptidecomposition, the aminogram and the mineral composition of the activeingredient are identical to those of Example 1.

Example 3 Serum Regrows Hair

An exemplary composition of the invention in serum form includes:

A. Water qs 100% Preservative 0.7% Propylene glycol  30% B. SimulgelFL10 (Seppic) 0.5% DC200 (Dow Corning)   8% C. Lactic acid qs pH 5 D.Active ingredient EXAMPLE 2: 0.3%

The pH of the composition is 5. It is in the form of an opalescentliquid, without odor.

The serum of Example 3 has the following characteristics: sprayablelotion, fresh and silky application, slippery effect, immediatepenetration, soft and dry finish, smoothing effect on the hair.

It can be obtained by implementing the following steps:

-   -   Mix A and heat in a water bath at 50° C., making sure to        disperse the preservative well,    -   Cool with stirring,    -   Add B, while stirring and adjust the pH with C,    -   Add D with stirring at 1,500 rpm and check the complete        homogenization of the formula.

Example 4 Mask Regrows Hair

An example of a composition of the invention in the form of a mask forthe hair, is constituted by:

A. Water qs 100% Preservative 0.7% Propylene glycol  20% Glucose acetate  6% B. Active ingredient EXAMPLE 2: 0.3% C. Simulquat HC305 (Seppic)  6%

The pH of the composition is 4.5.

It is in the form of a thick and compact white gel.

The mask of Example 3 has the following characteristics: firm grip, easyapplication, film-forming effect, easy rinsing, soft and dry finish.

It can be obtained by implementing the following steps:

-   -   Mix A and heat in a water bath at 50° C., making sure to        disperse the preservative well,    -   Add B with stirring and ensure good homogenization,    -   Add C with vigorous stirring (3,000 rpm) and stir until complete        homogenization of the gel.

Example 5 Hair Regrowth Treatment

An example of a composition of the invention in the form of treatmentfor the hair, is constituted by:

A. Water — qs 100% Preservative — 0.7% Dub Diol (Stéarinerie  20%DUBOIS) B. Satiaxane CX930 (Cargill) 0.1% Satialgine US551 (Cargill)  3% C. Active ingredient EXAMPLE 1: 0.7% D. GENUGEL carrageenan CG- (CPKelco) 2.5% 130 Lauroyl Lysine (HYTECH   2% Laboratory) D-panthenol(XINFU)   1% Phytosqualane (Sophim)   1%

The pH of the composition is 6.1.

It is in the form of a thick, smooth, odorless, unbleached gel.

The treatment of Example 5 has the following characteristics: Gentlegrip, soft and even application on the skin and hair, cool andmoisturizing effect, easy rinsing, smooth and soft finish.

It can be obtained by implementing the following steps:

-   -   Mix A and heat in a water bath at 50° C., making sure to        disperse the preservative well,    -   Add B, stirring and ensure good homogenization of the gels,    -   Add C, and check the good dispersion of the active ingredient,    -   When cold, add D in the order indicated and stir (2,500 rpm)        until complete homogenization.

Example 6 Treating Washing Base

An example of a composition of the invention in the form of treatmentfor the hair, is constituted by:

A. Oramix LS30 (Seppic) 25% Betadet S20 (Kao Corporation) 25% OramixNS20 (Seppic) 20% Somepon T25 (Seppic)  4% B. Preservative —  1%Carbopol Ultrez10 (Noveon) 0.1%  C. ACTIVE INGREDIENT EXAMPLE 1: 0.7% D. NaOH — qs pH 4.8 E. Dub Grenn OD4 (Stéarinerie DUBOIS) 15% Baobab Oil(Sophim)  5% Biophytosebum (Sophim)  5%

The pH of the composition is 5.

It is in the form of a liquid gelled base, slightly unbleached,odorless.

The washing base of Example 6 has the following characteristics: fluidflow, soft touch, extremely strong foaming power, good water dispersionwith creation of a soft, white and compact foam, easy rinse, veryslightly film-forming soft finish, moisturizing effect.

It can be obtained by implementing the following steps:

-   -   Mix A without stirring to avoid the formation of foam,    -   Add B, then C and stir gently, making sure that the powders are        well dispersed.    -   Adjust the pH with D and with more intense stirring (1000 rpm),        add the fatty phase and stir until the gel is completely        homogenized.

Example 7 Treatment Lotion

An example of a composition of the invention in the form of a lotion forhair, includes:

A. Water qs 100% Preservative 0.7% Propylene glycol   8% Activeingredient EXAMPLE 2 0.3% B. Glycerol 2.0% C. Alcohol 10.0% 

It is in the form of an ethanolic lotion, transparent liquid.

It can be obtained by implementing the following steps:

-   -   Mix A without stirring to avoid the formation of foam,    -   Add B, then C, and stir gently, ensuring good dispersion.

Evaluation of the Cosmetic Efficacy of the Invention I. In Vitro Tests

1 Effect of a Hydrolysate of Pichia Minuta of the Invention on theMitochondrial Dynamics Necessary for the Growth of Hair Follicles

The objective of this study is to evaluate the effect of an activeingredient of the invention on the mitochondrial dynamics, an elementnecessary for the growth of hair follicles.

For this, the synthesis of mitofusin 1 was evaluated. Mitofusin 1 is amajor player in mitochondrial fusion in human fibroblasts of the dermalpapilla. When hair growth is encouraged, mitochondrial dynamics aremodified. This results in an increase in the proportion of large fusedmitochondria compared to small fissioned mitochondria in dermal papillafibroblasts. This state is favorable for energy production.

The study was carried out by Western blot pursuant to the operatingprocedure described below.

On day 0, the human fibroblasts of the dermal papilla are inoculatedinto culture medium and incubated at 37° C.

On day 3, the cells are treated: the fibroblasts are treated with mediumeither containing or not the active ingredient at 0.02% and 0.04% (finalV/V).

On day 5, the cell extracts are recovered and then stored at −80° C.waiting for assay by Western blot in accordance with the followingcharacteristics:

-   -   electrophoresis is carried out on pre-cooled gel and the        proteins are then transferred on a PVDF membrane,    -   immunolabeling is carried out using a primary anti-mitofusin 1        antibody,    -   the bands are visualized, semi-quantified by densitometry with a        CCD camera, then analyzed using software.

The results are shown in Table 5.

TABLE 5 Effect of hydrolysate of Pichia minuta on mitofusin 1 synthesisby dermal papilla fibroblasts Mitofusin 1 level Efficiency/Control (UA)(%) Control 0.929 Active ingredient - 1.180 27 Example 2 at 0.02% ActiveIngredient - 1.412 52 Example 2 at 0.04%

These results show that tested at 0.04%, a hydrolysate of Pichia minutasignificantly increases mitofusin 1 synthesis by 52% in humanfibroblasts of the dermal papilla. Thus, it acts favorably on themitochondrial dynamics, a key element for the production of the energynecessary for the growth of hair follicles.

2. Effect of a Hydrolysate of Pichia Minuta of the Invention on theRegulation of “Signal” Molecules Described in Androgenetic Alopecia

The objective of this study is to evaluate the effect of an activeingredient of the invention on the regulation of “signal” moleculesdescribed in androgenetic alopecia.

For this, the study included evaluating dermal papilla fibroblaststreated with a solution of DHT mimicking alopecia:

-   -   IL-6 (interleukin 6), a cytokine secreted by human fibroblasts        of the dermal papilla which inhibits hair growth;    -   DKK1 (Dickkopf 1), an inhibitor of the Wnt/β-catenin pathway, a        major player in alopecia;    -   P16, a cell cycle protein associated with premature ageing of        alopecia zone dermal papilla fibroblasts.

This study was carried out by ELISA and quantitative PCR, according tothe operating protocol described below.

On day 0, the human fibroblasts of the dermal papilla are inoculatedinto culture medium and incubated at 37° C.

On day 3, the fibroblasts are treated with a solution of DHT containingor not the active ingredient at 0.02% and 0.04% (final V/V).

On day 5, at the end of the incubation, the cell supernatants arerecovered. The IL-6 assay is performed using an ELISA assay kit. TheRNAs were reverse-transcribed and the resulting complementary DNAs wereanalyzed by the quantitative PCR technique.

Expression of DKK1 and P16 mRNAs was also studied. In parallel, mRNAs ofRibosomal Protein S27 (RPS27), Hypoxanthine Phosphoribosyltransferase 1(HPRT1), beta-Glucuronidase (GUSB) were analyzed as internal referencecontrols for standardization.

Quantification of fluorescence incorporation (SYBR Green) iscontinuously measured using a thermal cycler. The analysis of Ct(relative quantification) is performed using software.

The results are shown in Tables 6 and 7.

TABLE 6 Effect of a hydrolysate of Pichia minuta on the release ofinterleukin 6 by dermal papilla fibroblasts in a normal or alopecicmodel. IL-6 level Ability to limit the (fg/μg of protein) secretion ofIL-6 (%) Normal model Control 1,284 Active ingredient - Example 2 1,1820.04% Alopecic model Control 1,736 Active ingredient - Example 2 1,43167 0.02% Active ingredient - Example 2 1,299 97 0.04%

TABLE 7 Effect of a hydrolysate of Pichia minuta on the expression ofDKK1 and P16 by dermal papilla fibroblasts in a normal or alopecicmodel. Ability to Ability to limit the limit the DKK1 expression of P16expression of (%) DKK1 (%) (%) P16 (%) Normal model Control 100 100Active ingredient - 92 105 Example 2 0.04% Alopecic model Control 212195 Active ingredient - 132 71 139 59 Example 2 0.02% Activeingredient - 110 91 128 71 Example 2 0.04%

It is found that DHT significantly increases IL-6 secretion andexpression of DKK1 and P16 by human fibroblasts of the dermal papilla,thus mimicking the expression modification of “signal” moleculesdescribed in androgenetic alopecia.

Tested at 0.04%, the active ingredient of the invention makes itpossible to significantly limit the secretion of IL-6 by 97% and theexpressions of DKK1 and P16 by 91% and 71%, respectively.

Thus, it corrects the abnormal production of “signal” molecules involvedin androgenetic alopecia.

3. Effect of a Hydrolysate of Pichia Minuta of the Invention on theEpigenetic Modifications Associated with Androgenetic Alopecia

The objective of this study is to evaluate the effect of an activeingredient of the invention on the epigenetic modifications associatedwith androgenetic alopecia.

For this, the expression of miRNA in a model of human dermal papillafibroblasts, treated with a solution of dihydrotestosterone (DHT) (inorder to mimic alopecia) was evaluated. This study focuses on two miRNAsinvolved in hair growth: 3663-3p and let-7a-3p whose expressions areinduced by DHT. Among the biological pathways targeted by these miRNAsis the Wnt/βcatenin signaling pathway which is essential for hairgrowth.

The study was carried out by quantitative PCR, according to thefollowing operation protocol.

On day 0, the human fibroblasts of the dermal papilla are inoculated inculture medium and incubated at 37° C. in an atmosphere containing 5%CO₂.

On day 3, the fibroblasts are treated with a solution of DHT which doesor does not contain the active ingredient of the invention at 0.02% and0.04% (final V/V).

On day 4, the RNAs were reverse-transcribed and the resultingcomplementary DNAs were analyzed by quantitative PCR technique.

The expression of microRNAs: 3663-3p and let-7a-3p has been studied.

In parallel, small nucleolar RNA C/D box 68 (SNORD68) was analyzed as aninternal reference control for standardization.

Quantification of fluorescence incorporation is measured continuouslyusing a thermocycler. The analysis of the Ct (relative quantification)is performed using software.

The results are shown in Table 8.

TABLE 8 Effect of a hydrolysate of Pichia minuta on the expression of3663-3p and let-7a-3p miRNAs by dermal papilla fibroblasts in a normalor alopecia model. Ability to Ability to limit limit the miRNA theexpression expression of miRNA 3663-3p of miRNA miRNA let- let-7a-3p (%)3663-3p (%) 7a-3p (%) (%) Normal model Control 100 100 Activeingredient - 86 120 Example 2 0.04% Alopecic model Control 172 172Active ingredient - 143 40 125 65 Example 2 0.02% Active ingredient -136 50 108 89 Example 2 0.04% Active ingredient - 133 54 Example 1 0.04%

DHT induces expression of 3663-3p and let-7a-3p miRNAs in human dermalpapilla fibroblasts, mimicking the epigenetic changes associated withandrogenetic alopecia.

It is found that, tested at 0.04%, a hydrolysate of Pichia minutasignificantly reduces the expression of miRNA 3663-3p and let-7a-3p byrespectively 50% and 89%.

Thus, an active ingredient of the invention makes it possible to limitthe epigenetic modifications associated with androgenetic alopecia.

4. Effect of a Hydrolysate of Pichia Minuta of the Invention on theActivity of the Dermal Papilla in Androgenetic Alopecia

The objective of this study is to evaluate the effect of an activeingredient of the invention on the activation of the dermal papilla.

For this, the versican synthesis was measured. It is a proteoglycan thatis specific to the growth phase. The evaluation was performed in a 3Dmodel of fibroblasts in the form of spheroids, treated with a solutionof DHT. This model mimics the inactivation of the dermal papilla, afactor contributing to the lack of growth associated with androgeneticalopecia.

The study was performed by immunohistological staining according to thefollowing operating protocol.

On day 0, the human fibroblasts of the dermal papilla are inoculatedinto culture medium and incubated at 37° C.

On day 1, the spheroids formed are treated with a solution of DHT whichdoes or does not contain the active ingredient of the invention at 0.04%(final V/V).

On day 4, the spheroids are recovered and secured.

Immunohistological labeling of versican is performed. Visualization isperformed on a confocal microscope coupled to an image analysis system.

Versican synthesis is proportional to the intensity of fluorescence(green color) present on the spheroids.

The nuclei of the cells appear to be blue in color.

The results being qualitative, we defined 3 levels of synthesis ofversican:

-   -   Low synthesis +    -   Average synthesis ++    -   Strong synthesis +++

The results are given in Table 9.

TABLE 9 Effect of a hydrolysate of Pichia minuta on the synthesis ofversican in dermal papilla fibroblasts spheroids in an alopecic model.Synthesis of versican Normal model Control +++ Alopecic model Control +Active principle - example 2 ++ 0.04%

DHT induces a decrease in the synthesis of versican, in a model of humanfibroblasts of dermal papilla in the form of spheroids, mimicking aninactivation of the dermal papilla induced during androgenetic alopecia.

It is found that when tested at 0.04%, the active ingredient of theinvention stimulates the synthesis of versican in this model. Thus, ithelps maintain the activity of the dermal papilla and its ability toinduce the growth of hair follicles.

5. Effect of a Hydrolysate of Pichia Minuta of the Invention on theGrowth of Hair Follicles in Androgenetic Alopecia

The objective of this study is to evaluate the ability of an activeingredient of the invention to maintain hair follicles in a growth phase(anagen phase).

For this, we conducted a study on the ex vivo hair follicle culturemodel developed by Philpott et al. treated with a solution of DHT inorder to mimic the growth reduction associated with androgeneticalopecia. This ability was evaluated by measuring:

-   -   the elongation of hair follicles;    -   the synthesis of versican and Ki-67, two key proteins of hair        growth.

The evaluation of the elongation is carried out by measuring the size ofhair follicles after photographs. The syntheses of versican and Ki-67are studied by immunohistological labeling. The operating protocol isdescribed hereafter.

On day 0, the hair follicles are obtained from scalp fragments andincubated in culture medium at 37° C.

On day 1, the follicles are photographed and measured on a microscopecoupled to an image analysis system.

The follicles selected (in the anagen phase) are then treated with asolution of DHT which does or does not contain the active ingredient ofthe invention at 0.02% and 0.04% (final V/V).

On day 5, two studies are carried out: the study of the elongation ofhair follicles and the study of synthesis of Versican and Ki-67.

Study of the Elongation of Hair Follicles

The follicles are photographed and measured on a microscope coupled toan image analysis system. The elongation of each follicle is equal toits growth between day 0 and day 5.

The follicles are treated with a solution of DHT containing or not theactive ingredient at 0.02% and 0.04% (final V/V).

Study of the Synthesis of Versican and Ki-67

The follicles are recovered and included in Tissue-Tek® and then frozen.Sections (4 μm) are then made using a cryostat Immunohistologicallabeling of versican and Ki-67 is performed. Visualization is performedon a confocal microscope coupled to an image analysis system.

Versican synthesis is proportional to the intensity of fluorescence(green color) present on sections of hair follicles.

The synthesis of Ki67 is proportional to the intensity of thefluorescence (yellow color) present on sections of hair follicles.

On day 7, the follicles are again photographed and measured. Theelongation of each follicle is equal to its growth between day 0 and day7.

The results of the effect on the elongation of hair follicles are givenin Table 10.

TABLE 10 Effect of a hydrolysate of Pichia minuta on the growth of hairfollicles ex vivo in an alopecic model. Elongation Ability to restore(μm) elongation (%) Day 0-day 5 Normal model Control 1,201 Alopecicmodel Control 743 Active principle - 822 +11 Example 2 0.02% Activeingredient - 887 +31 Example 2 0.04% Day 0-day 7 Normal model Control1,617 Alopecic model Control 961 Active principle - 1,038 +12 Example 20.02% Active ingredient - 1,206 +37 Example 2 0.04%

It is found that DHT significantly reduces the growth of hair folliclesat day 5 and day 7 by −31% and −37%, respectively. As a result, DHTmimics the lack of hair growth associated with androgenetic alopecia.

Tested at 0.04%, an active ingredient of the invention makes it possibleto significantly increase the growth of hair follicles on day 5 and day7 by respectively +31% and +37%.

The results of the effect on hair matrix of hair follicles are given inTable 11.

The results being qualitative, 3 synthesis levels of versican and Ki-67were defined:

-   -   Low synthesis +    -   Average synthesis ++    -   Strong synthesis +++

TABLE 11 Effect of a hydrolysate of Pichia minuta on the synthesis ofversican and Ki-67 by ex vivo hair follicles in an alopecic model.Synthesis of Versican and Ki-67 Normal model Control +++ Alopecic modelControl + Active ingredient - Example 2 ++ 0.04%

It is found that DHT significantly reduces the synthesis of versican andKi-67. As a result, DHT mimics the metabolic alterations associated withandrogenetic alopecia.

Tested at 0.04%, an active ingredient of the invention makes it possibleto maintain a significant synthesis of versicans, and the proliferativepower of keratinocytes.

Thus, the use of the invention makes it possible to limit the reductionin growth associated with androgenetic alopecia and keeps hair folliclesin an anagen phase.

II. In Vivo Tests

The volunteers participating in the in vivo tests were selectedaccording to several criteria. These are men under 55 years of age, withmild to moderate alopecia and a minimum of 20% hair in the telogenphase.

1 Effect of a Hydrolysate of Pichia Minuta of the Invention on CapillaryGrowth

The objective of this study was to evaluate, in vivo, the anti-hair losseffect of an active ingredient of the invention (Example 2) formulatedat 0.3% in a lotion (Example 7), in volunteers with mild to moderatealopecia.

This study was conducted on 22 volunteers aged between 31 and 55 (meanage 43±8 years), having applied 1 ml of the lotion of the inventionevery morning and evening for 8 months.

The anti-hair loss effect was studied by the phototrichogram method frommeasurements taken at time 0, 3, 6 and 8 months after the start oftreatment. The following parameters were analyzed:

-   -   capillary density,    -   quantity of hair in the anagen phase: indicator of hair growth;    -   quantity of hair in telogen phase: indicator of hair loss;    -   Anagen/Telogen ratio: hair growth coefficient.

A summary of results on capillary density is presented in Table 12.

TABLE 12 Effect of a hydrolysate of Pichia minuta formulated at 0.3% ofcapillary density after 3, 6 and 8 months of twice-daily application.Variation/day 0 (%) 3 months +21.3 6 months +22.1 8 months +24.9

It is found that an active ingredient of the invention, formulated at0.3% increases the capillary density already after 3 months ofapplication (+21.3%). This effect is prolonged and intensified after 6and 8 months of treatment (respectively +22.1 and +24.9%).

A summary of results on hair growth is presented in Table 13.

TABLE 13 Effect of a hydrolysate of Pichia minuta formulated at 0.3% ofhair growth after 3, 6 and 8 months of twice-daily application.Variation/Day one (%) Improvement of the Hair in growth phaseAnagen/Telogen ratio (anagen phase) (%) (%) 3 months +24.7 +73.1 6months +38.7 +85.7 8 months +49.1 +112.7

It is found that the active ingredient of the invention shows an actionon capillary growth, already after 3 months of application, whichintensifies during treatment.

This results in a statistically significant increase in the proportionof hair in the anagen phase. In addition, there is a significantincrease in the Anagen/Telogen ratio.

A summary of the results on the reduction of the quantity of hair incapillary telogen phase is presented in Table 14.

This parameter, characteristic of a reduction in hair loss, wasevaluated by phototrichogram.

TABLE 14 Effect of a hydrolysate of Pichia minuta formulated at 0.3% ofhair loss in the telogen phase after 3, 6 and 8 months of twice-dailyapplication. Variation/Day one (%) 3 months −14.0 6 months −121.9 8months −126.1

Under the conditions of this study, the active ingredient of theinvention formulated at 0.3% in a lotion significantly reduces theproportion of hair in the telogen phase after 3 months of application(−14.0%). This effect intensifies after 6 and 8 months of treatment(respectively −21.9% and −26.1%).

These different results are illustrated in FIGS. 5A/6A (at day 0 beforeapplication) and 5B/6B (after 8 months of application). The total hairgain on the scalp between day 0 and 8 months for this illustration is74,160 hairs.

By stimulating hair growth (anagen phase) and decreasing the proportionof hair in the telogen phase, the use of an active ingredient of theinvention makes it possible to strengthen and redensify the hair.

2. Effect of a Hydrolysate of Pichia Minuta of the Invention on HairLoss

The objective of this study was to evaluate, in vivo, the ability of anactive ingredient of the invention (Example 2) formulated at 0.3% in alotion (Example 7), to curb the hair loss of volunteers with mild tomoderate alopecia.

This study was performed on 22 volunteers aged between 31 and 55 (meanage 43±8 years), having applied 1 ml of the lotion of the inventionevery morning and evening.

The magnitude of hair loss was studied using the wash test methodperformed before starting and 3, 6 and 8 months after the start oftreatment.

A summary of the results corresponding to the effect of an activeprinciple of the invention formulated at 0.3% on the hair loss evaluatedby means of counting after wash testing is presented in Table 15.

TABLE 15 Effect of a hydrolysate of Pichia minuta formulated at 0.3% ina lotion on hair loss after 3, 6 and 8 months of twice-dailyapplication. Variation/Day one (%) 3 months −121.2% 6 months −130.9% 8months −134.0%

Under the conditions of this study, already after 3 months oftwice-daily applications, the composition of the invention slows hairloss by reducing the number of hairs recovered after shampooing by21.2%.

This effect is prolonged and intensified after 6 months of treatment toachieve an average decrease in hair loss of 30.9%, the maximum decreasebeing 81%. More than 8 out of 10 volunteers presented this effect.

Finally, after 8 months of treatment, the invention stabilizes hair lossby limiting the number of hairs lost by 34.0%.

Comparative Tests: Comparison of the Active Principle of the Inventionwith Other Hydrolysates of Yeast

Comparative Test 1

The purpose of this test is to show the importance of the choice ofyeast.

An identical enzymatic hydrolysis process was implemented on 3biomasses:

-   -   a Pichia minuta biomass    -   a Pichia heedii biomass    -   a Saccharomyces cerevisiae biomass

This method included the implementation of the following steps:

-   -   solubilization of biomass in water at 100 g/L,    -   enzymatic hydrolysis using a protease,    -   enzymatic inactivation thermally at 80° C. for 1 hour,    -   decantation to remove the insoluble phase, recover supernatant,    -   filtration, and recovery of the filtrate.

The analytical characteristics of the products obtained are presented inthe table below:

TABLE 16 Analytical characteristics of the three hydrolysates of yeastHydrolysate of Hydrolysate of Hydrolysate of Saccharomyces Pichia minutaPichia heedii cerevisiae Dry matter (MS) g/L 36 22 24 pH 5.4 5.5 5.2Color L, a, b 99.48 99.42 99.97 −10.78 −11.18 −10.27 3.29 3.95 0.78 Ashg/L 7.8 4.6 4.1 % ash/MS 21.7% 20.9% 17.1% Protein (Lowry 18.6 6.8 1.5method) g/L % Protein/MS 52% 31% 6%

It can be seen that these hydrolysates do not have the same analyticalcharacteristics, especially as regards the protein content, which arevery different for the three products obtained from the same process,but from different yeasts.

The size of the proteins is also different, as can be seen in thechromatogram of the proteins of the three products, as shown in FIG. 3.

These 3 hydrolysates were then tested for their effect on the expressionof P16, the tests having been carried out according to the conditionsand the protocol described in point I.2 of the evaluation part of thecosmetic effect of the present application.

The results obtained are shown in Table 17.

TABLE 17 Ability of the three hydrolysates to inhibit P16. Ability tolimit the P16 (%) expression of P16 (%) Normal model Control 100Alopecic model Control 195 Hydrolysate of Pichia minuta 0.04% 128 71Hydrolysate of Pichia heedii 1% 222 0 Hydrolysate of Saccharomyces 214 0cerevisiae 1%

These results clearly show that the choice of yeast is important, sincea same production process of the active ingredient applied to 3 yeastbiomasses of the same family, leads to three different products, onlyone of which is effective on a key parameter in fighting hair loss.

Comparative Test 2

The aim of this test is to compare the effectiveness of a cosmeticactive ingredient, a hydrolysate of Pichia anomala described in patentFR3016521, with that of the active ingredient of the invention in orderto show that a cosmetic active ingredient derived from a yeast of thegenus Pichia, does not necessarily work.

The analytical characterization of the two active ingredients ispresented in Table 18.

TABLE 18 Analytical characteristics of both hydrolysates Hydrolysate ofHydrolysate of Pichia minuta Pichia anomala (FR3016521) % Ash/MS 26%34.7% % Protein/MS 59%  59% % sugars/MS 15%  6.3%

It is found that these hydrolysates have similar analyticalcharacteristics, especially with regard to the protein contents whichare identical.

The characterization of the protein fraction is presented in Table 19.

TABLE 19 Characterization of the protein fraction of the twohydrolysates Hydrolysate of Pichia Hydrolysate anomala of Pichia(FR3016521) minuta Fraction 1: 13.0% Fraction 1: 0.3% Mm > 5000 Da Mm >10,000 Da Fraction 2: 2000 < 38.4% Fraction 2: 3500 < 2.9% Mm ≤ 5000 DaMm ≤ 10 000 Da Fraction 3: 2000 < 10.5% Mm ≤ 3500 Da Fraction 3: 243 <46.3% Fraction 4: 243 < 83.3% Mm ≤ 2000 Da Mm ≤ 2000 Da Fraction 4: 0 <2.3% Fraction 5: 0 < 3.0% Mm ≤ 243 Da Mm ≤ 243 Da

Protein size is also similar, since both hydrolysates contain 59%protein, more than 87% of which have a molecular weight of less than 5kDa.

These 2 hydrolysates were then tested for their effect on the expressionof P16, the tests having been carried out according to the conditionsand the protocol described in point I.2 of the evaluation part of thecosmetic effect of the present application.

The results obtained are shown in Table 20.

TABLE 20 Ability of both hydrolysates to inhibit P16. Ability to limitthe P16 (%) expression of P16 (%) Normal model Control 100 Alopecicmodel Control 195 Hydrolysate of Pichia minuta 0.04% 128 71 Hydrolysateof Pichia anomala 1% 225 0

These results still show that it is obvious that the choice of yeast isimportant, since a hydrolysate of Pichia anomala from a yeast of thePichia genus, also having the same protein content as the hydrolysate ofPichia minuta to which it is compared, does not show efficacy on thechosen capillary marker.

Comparative Test 3

The aim of this test is to compare the effectiveness of a hydrolysate ofTorulaspora delbrueckii, described in patent FR2979541, with that of theactive ingredient of the invention of Example 1, in order to show that acosmetic active ingredient derived from a yeast having a similaranalytical characteristic, does not necessarily work.

The analytical characterization of the two active ingredients ispresented in Table 21.

TABLE 21 Analytical characteristics of both hydrolysates Hydrolysate ofTorulaspora Hydrolysate of delbrueckii Pichia minuta (FR2979541) %ash/MS 26% 28.3% % Protein/MS 59% 64.3% % sugars/MS 15% 6.95% %Polyphenols/MS  0% 0.25%

It is found that these hydrolysates have similar analyticalcharacteristics, particularly with regard to the protein contents whichare close.

The characterization of the protein fraction is presented in Table 22.

TABLE 22 Characterization of the protein fraction of the twohydrolysates Hydrolysate of Torulaspora Hydrolysate delbrueckii ofPichia (FR2979541) minuta Fraction 1: 3.3% Fraction 1: 0.3% Mm > 5000 DaMm > 10,000 Da Fraction 2: 2000 < 25.64% Fraction 2: 3500 < 2.9% Mm ≤5000 Da Mm ≤ 10 000 Da Fraction 3: 2000 < 10.5% Mm ≤ 3500 Da Fraction 3:243 < 68.29% Fraction 4: 243 < 83.3% Mm ≤ 2000 Da Mm ≤ 2000 Da Fraction4: 0 < 2.77% Fraction 5: 0 < 3.0% Mm ≤ 243 Da Mm ≤ 243 Da

Protein size is also similar, since both hydrolysates contain more than93% having a molecular weight of less than 5 kDa.

These 2 hydrolysates were then tested for their effect on the expressionof P16, the tests having been carried out according to the conditionsand the protocol described in point I.2 of the evaluation part of thecosmetic effect of the present application.

The results obtained are shown in Table 23.

TABLE 23 Ability of both hydrolysates to inhibit P16. Ability to limitthe expression P16 (%) of P16 (%) Normal model Control 100 Alopecicmodel Control 195 Hydrolysate of Pichia minuta 0.04% 128 71 Hydrolysateof Torulaspora delbrueckii 1% 201 0

These results further show that the choice of yeast is important, sincea hydrolysate of another yeast having a similar analyticalcharacterization (protein/ash/sugar content) and identical protein sizes(93% of proteins<5000 Da), has no efficacy on the chosen capillarymarker.

Comparative Test 4

The objective of this test is to demonstrate that a hydrolysate ofPichia minuta does not have the same analytical characteristics and istherefore different from a Pichia minuta biomass, as it exists innature.

First, the hydrolysate does not contain live yeasts whereas the biomasscontains them.

The method of microorganism culture on agar, allows this verification.It has been used for the hydrolysate of Example 1 on the one hand andfor a biomass of Pichia minuta on the other hand:

-   -   Isolation by dial streak at the angle of 1 μL,    -   Culture medium: Sabouraud    -   Incubation 48 h at 30° C.

The results are shown in FIG. 4B for the hydrolysate and in FIG. 4A forthe biomass.

No growth is not observed (FIG. 4B) on the agar with the hydrolysate ofexample 1, whereas on the other hand (FIG. 4A) a growth of white, round,shiny, oily colonies with a diameter of less than 0.5 mm is observed onagar with Pichia minuta biomass.

The analytical characterization of the hydrolysate of Example 1 and abiomass of Pichia minuta is further presented in Table 24.

TABLE 24 Analytical characteristics of the hydrolysate of the inventionand of a biomass of Pichia minuta Hydrolysate of Pichia minuta Biomassof (Example 1) Pichia minuta % ash/MS 26%  8% % Protein/MS 59% 41% %sugars/MS 15% 36% Not determined — 15%

These results show that there are important analytical differencesbetween biomass and hydrolysate of Pichia minuta. Biomass is the naturalraw material on which the enzymatic hydrolysis process is applied toobtain the active ingredient of the invention, hydrolysate of Pichiaminuta.

While at least one exemplary embodiment of the present invention(s) isdisclosed herein, it should be understood that modifications,substitutions and alternatives may be apparent to one of ordinary skillin the art and can be made without departing from the scope of thisdisclosure. This disclosure is intended to cover any adaptations orvariations of the exemplary embodiment(s). In addition, in thisdisclosure, the terms “comprise” or “comprising” do not exclude otherelements or steps, the terms “a” or “one” do not exclude a pluralnumber, and the term “or” means either or both. Furthermore,characteristics or steps which have been described may also be used incombination with other characteristics or steps and in any order unlessthe disclosure or context suggests otherwise. This disclosure herebyincorporates by reference the complete disclosure of any patent orapplication from which it claims benefit or priority.

1. A cosmetic active ingredient comprising: a hydrolysate of Pichiaminuta.
 2. The cosmetic active ingredient according to claim 1, whereinthe hydrolysate of Pichia minuta comprises peptides.
 3. The cosmeticactive ingredient according to claim 2, wherein at least 50% by weightof the peptides of the hydrolysate are peptides having a molecularweight of less than 3500 Da.
 4. The cosmetic active ingredient accordingto claim 2, wherein the peptides comprise peptides wtih a molecularweight of less than 3500 Da and the peptides with a molecular weight ofless than 3500 Da represent at least 40% of a weight of a total drymaterial of the hydrolysate.
 5. The cosmetic active ingredient accordingto claim 1, wherein the hydrolysate comprises at least 5% of peptides byweight of a dry matter of the hydrolysate.
 6. The cosmetic activeingredient according to claim 1, wherein the hydrolysate furthercomprises at least one mineral.
 7. The cosmetic active ingredientaccording to claim 1, wherein the cosmetic active ingredient is aliquid.
 8. The cosmetic active ingredient according to claim 1, whereinthe hydrolysate comprises an enzymatic or chemical hydrolysate.
 9. Thecosmetic active ingredient according to claim 1, wherein the cosmeticactive ingredient comprises a solid and further comprising anatomization carrier selected from the group consisting of: maltodextrin,gum arabic, soy lecithin, and isomalt.
 10. The cosmetic activeingredient according to claim 9, wherein the hydrolysate of Pichiaminuta represents between 25 and 75% of the weight of the activeingredient.
 11. A non-therapeutic method for fighting hair loss andactivating its regrowth; comprising: forming a cosmetic activeingredient according to claim 1; and, applying the cosmetic activeingredient to a hair or a scalp of an individual.
 12. (canceled) 13.(canceled)
 14. A cosmetic composition comprising: at least 0.05% byweight of a cosmetic active ingredient in liquid form according toclaim
 1. 15. The cosmetic composition according to claim 14, wherein thecosmetic composition is in the form of a lotion, shampoo, cream, ormousse.
 16. A non-therapeutic cosmetic method for fighting hair loss andpromoting its regrowth, comprising: forming a cosmetic compositionaccording to claim 14; and, applying the cosmetic composition to a hairor a scalp of an individual.
 17. A non-thcrapcutic cosmetic method forfighting hair loss and promoting its regrowth, comprising: forming acosmetic composition according to claim 14; and, applying the cosmeticcomposition to a hair or a scalp of an individual at least once a dayfor at least one month.